The present invention relates to products and methods used in flow cytometry instrumentation for the analysis of biological particles or cells. More particularly, the invention relates to synthetic polymer materials and methods for standardizing or calibrating flow cytometry instruments prior to using the instruments for analyzing such particles, for example, the cellular components of whole blood.
The analysis of particles, particularly biological particles, and cells is routinely performed using a variety of commercially-available instruments which determine the characteristics of such particles based on one or more light-related signals which pass through the instrument. Flow cytometers allow the determination of the characteristics of particles using techniques in which the particles are moving in a liquid stream or carried in a suspension. Typically, in flow cytometry instruments, cells or other biological particles flow in a liquid stream so that each particle, virtually one cell at a time, passes through a sensing region that is capable of measuring the physical or chemical characteristics of the particles.
A variety of signals associated with different characteristics of the particles under analysis may be detected. Such signals include electrical, acoustical, optical and radioactive. Flow cytometers generally rely on optical signals for the analysis of particles which pass through the instrument. Whether or not an instrument analyzes particles in a static or a dynamic state, those skilled in the art will appreciate that calibration and standardization are required prior to performing particle analyses. Under normal circumstances, calibration occurs as one or more preliminary presteps in preparing instruments for proper use and measurement and to ensure accurate and reliable assay results. This is especially important since cells, or other biological particles, are extremely small and the signals to be detected, in relation to the size of the cells or particles, are often at a low magnitude.
Flow cytometers and other biological particle and cell analysis instruments are commonly calibrated with particles which simulate or approximate the types of particles or cells that are intended to undergo analysis. Thus, calibration particles should be selected or designed so that they have characteristics and parameters that are quite similar to those of the particles or cells to be tested in the instruments. Exemplary characteristics and parameters include similarities in size, volume, surface characteristics, granularity properties, and, if necessary, color features, such as stains, dyes, immunofluorescent tags, and the like. Accordingly, it is to be understood that the particles to be analyzed after calibration or standardization of an analyzer need not be restricted biological cells. For example, particles to be assayed may be found in oil-in-water suspensions, milk, or another nonbiological medium.
Hematology analyzers represent but one specific type of instrumentation designed and employed for the determination and measurement of the characteristics of cells and biological particles in whole blood. As a particular, yet nonlimiting, example, hematology systems commercially available from Bayer Corporation optically analyze, determine the characteristics of and measure erythrocytes (red blood cells), reticulocytes (immature red blood cells), leukocytes (white blood cells) and platelets in whole blood samples.
Past and present calibration procedures for flow cytometry instruments, including hematology analyzers, involve the use of fixed and/or sphered red blood cells, for example, human and chicken red blood cells, for the calibration steps and for standardizing optical signals such as light scattering. See, for example, U.S. Pat. No. 4,489,162 to Hawkins et al. and U.S. Pat. No. 4,777,139 to S.-C. Wong et al. While the use of red blood cells procured from mammals and humans may be reliable in some aspects of calibration procedures, there are drawbacks to the use of these types of xe2x80x9cnaturalxe2x80x9d calibrator cells. For example, the preparation of sphered and fixed red blood cells entails the blood-drawing process and other blood manipulations which involve contact with potentially biohazardous material. Further, because the stability of red cells as optical standards is limited, i.e., about six months, the cell standards must be reproduced periodically, typically, two or more times a year.
In addition to biological samples for calibration and standardization purposes, microspheres or microbeads have also been used for calibrating cellular analysis instruments. For example, U.S. Pat. No. 4,331,862 to W. L. Ryan describes beads composed of latex material for calibrating a particle counting instrument. Plastic microbeads are disclosed for calibrating flow cytometers and cell analysis instruments in U.S. Pat. No. 4,704,891 to D. J. Recktenwald et al. Calibration beads for calibrating flow cytometry instruments are commercially available from the Flow Cytometry Standards Corporation, Research Triangle Park, N.C. In general, such microsphere beads are not produced with a consideration for the proper refractive index values of actual cells and biological particles.
Nonbiological bead polymers having an average particle diameter of from 0.5 to 10 xcexcm and containing 1% to 60% of chemically bound fluorine are disclosed in U.S. Pat. No. 5,093,445 to W. Podszun et al. for use as matting agents and spacers in photographic recording materials. This patent does not relate to particle calibrators for flow cytometry and does not recognize refractive index as a critical parameter for such use. Indeed, the present inventors have determined that the polymer beads exemplified in this patent have refractive indices which are significantly higher than 1.45, which is considered to be at or near the upper limit of refractive index, particularly as it relates to blood cells, as described further hereinbelow.
Other types of flow cytometry instruments, such as fluorescence flow cytometers, use polymer beads made of materials such as polystyrene to standardize the optical signals. The signals typically include forward scatter (0-2 degrees), side scatter (90 degrees) and fluorescence intensity. The polymer bead materials, although safe and stable for about five years or more, are particularly unsatisfactory for light scattering flow cytometry instruments. More particularly, for particles of a given volume, currently-available polymer beads produce scattering signals that are different from those of red blood cells of the same volume, even if the red blood cells are sphered. This is because the refractive index (r.i.) of a polymeric material such as polystyrene is quite different from that of red blood cells. While the r.i. of polystyrene is high, i.e., about 1.59, the r.i. of a red blood cell is about 1.40-1.42. It is well known from the Mie Scattering Theory that the light scattering properties of small spheres are significantly influenced by their r.i. values. Thus, the scattering patterns of polymer beads currently used in flow cytometry instruments are not truly representative of the scattering patterns of actual red blood cells. Consequently, the forward and side scatter signals do not provide important information, such as cell size and refractive index, which are related to cell density and activation state.
In addition, platelet light scattering signals are currently standardized using either sphered and fixed red blood cells in the case of some types of analyzers, for example, commercial hematology analyzers, or using appropriately-sized polymer beads in the case of fluorescence flow cytometers. However, the scattering signals of platelets are typically much smaller than those of red blood cell (i.e., by more than a factor of 10). Also, the refractive index of platelets is only about 1.35 to about 1.40. Accordingly, the present invention advantageously provides a material with the light scattering properties associated with actual platelets, as well as with the stability and safety of non-biological polymeric materials for flow cytometry analyses.
Moreover, white blood cell light scattering signals are currently standardized using either sphered and fixed red blood cells or polymer beads. White blood cell scattering signals are typically larger than those of red blood cells and their refractive indices are lower, typically about 1.37 to about 1.40. Accordingly, the present invention advantageously also provides a material with the light scattering properties associated with actual white blood cells, as well as with the stability and safety of non-biological polymeric materials for flow cytometry analyses.
As a further particular example, some commercial hematology analyzers enumerate reticulocytes (immature red blood cells) based on a combination of their light scattering properties and their ability to bind to cationic dyes, such as Oxazine 750, and thus absorb light at the absorption wavelength of the dyes. Currently, the absorption channel is standardized using fixed and sphered red blood cells that do not contain dye. The standardization relies on the light scattered outside the collection cone-angle of the absorption detector to provide a xe2x80x9cpseudo-absorptionxe2x80x9d signal which mimics true light absorption. However, this method is not optimal, since the pseudo-absorption signal is significantly less than the true absorption signal produced by many reticulocytes. Thus, the optical standard used does not best represent true signals encountered in practice. Appropriately-colored polymeric beads made of materials such as polystyrene are also not useful for this purpose, since their high r.i. values result in scattering signals that are outside the scattering signal detector ranges. Therefore, for standardizing light scattering and absorption measurements of reticulocytes, it is desirable to produce a material that has the light scattering (and absorption) properties of reticulocytes and that is also safe and stable. In the case of fluorescence flow cytometry in which the combination of scattering and fluorescence properties is used to analyze blood cells, it is desirable to produce a material having the light scattering and fluorescence properties of the particular cells being analyzed, and having the safety and stability of non-biological polymeric materials.
Thus, there is a present need and a desire for standardization and calibration materials and procedures that provide accurate and reproducible results in the determination of the characteristics of both nonbiological and biological particles and cells, in a variety of analysis instruments, including hematology analyzers. There is also a current need for the production of safe and stable calibration particles for standardizing flow cytometry instruments. Such particles optimally need to have a long shelf-life and to have properties, such as particle size and refractive index, that are virtually identical to those of the natural particles and cells which they represent during the calibration/standardization process.
In addition, for hematology analyses, there is a need for the development of non-biological polymeric particles for standardizing flow cytometers wherein the standardizing polymer particles employed accurately portray all of the necessary parameters of the respective component blood cells to be analyzed following instrument calibration/standardization. The present invention is directed to the advantageous fulfillment of these current needs and goals in the art.
Accordingly, it is an object of the present invention to provide novel synthetic polymer particles for use in calibrating or standardizing a flow cytometer, particularly a hematology analyzer, prior to the analysis of nonbiological particles, or biological particles or cells, in which the calibrator/standardizing particles have virtually all of the optical and physical properties of actual particles or blood cells, e.g., authentic red blood cells, reticulocytes, platelets and/or white blood cells, to be analyzed, and the stability and safety of nonbiological polymeric materials.
In accordance with the present invention, the novel calibration particles are designed to have a narrow average particle diameter distribution, a well-defined average particle diameter and a well-defined refractive index value that are virtually identical to those of the authentic particles in various media, or cell types in a biological sample, such as a blood sample, which are to be analyzed.
Another object of the present invention is to provide novel reagents and methods for calibrating or standardizing a flow cytometry instrument prior to the analysis of biological particles or cells, particularly with regard to the analysis of the component cells in a whole blood sample.
Another object and advantage of the present invention is to provide calibration methods and materials that are substantial improvements over those that are known and used in the art. The invention provides the ability for those in the art to calibrate a particle analysis instrument by employing and relying on calibration standards (and/or thresholds) that have been predetermined and are easily manipulated and made available to the user. Using the standards and methods of the present invention during the standardization and calibration procedures of flow cytometry instruments allows for proper standardization and calibration of an instrument, proper alignment of the optical components of the instrument, and optimization of the performance of the instrument for subsequent particle and cell analysis, including the determination of cell size and refractive index from forward and side scatter measurements.
It is yet another object of the present invention to provide calibration/standardization methods and synthetic particles for use in several different particle analysis and detection systems that are based on a combination of light scatter as one measured parameter and the measurement of another known parameter, such as light scatter (i.e., scatter-scatter detection), electrical impedance (i.e., scatter-impedance detection), radio frequency (i.e., scatter-RF detection) and the like. It is to be further understood that the present invention and the advantages and features attendant thereto also may be utilized in other flow cytometry analysis instruments such as fluorescence cell sorters, and may extend to instruments such as image analyzers, automated microscopes, quantitative microscopes, fluorescence microscopes and the like. The materials and techniques of the present invention provide quality assurance for a variety of applications, including medical and clinical diagnostic applications for identifying and determining both normal and abnormal conditions.
Further objects and advantages afforded by the invention will be apparent from the detailed description hereinbelow.